KR20010006511A - Method of treating chronic progressive vascular scarring diseases - Google Patents

Abstract

The present invention is directed to preventing atherosclerosis diseases such as atherosclerosis, in particular to prevent the progression of chronic progressive vascular injury disease (CPVSD) or at least substantially reduce the rate of progression of the disease and to alleviate and / or alleviate previously formed lesions. A method of treating a suffering mammalian patient. The method of the present invention is to administer to a patient a pharmaceutical composition containing an effective amount of pentosan polysulfate (PPS) or a pharmaceutically acceptable salt thereof. Oral administration is preferred and the total daily dose of PPS or PPS salt is from about 5 to about 30 mg / kg patient weight or from about 350 to about 2,000 mg / day in adult patients.

Description

Treatment method of chronic progressive vascular injury disease {METHOD OF TREATING CHRONIC PROGRESSIVE VASCULAR SCARRING DISEASES}

1. Field of Invention

The present invention relates to pharmaceutical compositions and methods for use in the treatment of chronic progressive vascular injury diseases.

2. Description of Prior Art

Chronic Progressive Vascular Injury Disease (CPVSD) is a complication of the most common diseases in developed countries, including diabetes mellitus, hypertension, and various types of hyperlipidemia. The treatment modality of the present invention for CPVSD is to treat the cause. Unfortunately, in most cases there is no known treatment or very difficult to control it in the general population. Moreover, CPVSD is not only already known about when its causes are medically complications, but is further developing. Therefore, CPVSD is considered to be the most serious complication because it causes kidney failure, seizures, heart disease and blindness, and therefore complications must be treated.

In general, CPVSD is characterized by smooth muscle vascular cell changes. One of the major changes is an increase in the amount and form of connective tissue they synthesize. This change significantly reduces functionality. In the case of blood vessels, elasticity is lost, and contraction and relaxation do not occur, the wall of the blood vessel is thickened, and the lumen of the vessel is narrowed. As a result, blood flow is reduced or completely blocked. Examples of vascular injury diseases characterized by such pathophysiological processes include chronic progressive glomerular disease (eg, diabetes-derived glomerulosclerosis (injury)); Progressive renal failure after kidney transplantation; Occlusion of anastomosis used to enter and exit blood vessels of end-stage renal disease patients undergoing hemodialysis; Other chronic small vessel diseases (eg, diseases seen in hypertensive patients); Relapse of stenosis in patients undergoing coronary bypass surgery; And diabetic retinopathy.

The purpose of all treatments for CPVSD is to reduce the excess extracellular matrix (damage) already formed to restore normal vascular openness and function or to prevent further progression or substantially reduce the rate of progression. However, despite the urgent need for the treatment of chronic progressive diseases, no direct methods for controlling connective tissue synthesis or abnormalities of smooth muscle tissue metabolism have been developed. It has been believed that this disease cannot be prevented or reversed.

Therefore, it is particularly important to develop a regimen of therapies for CPVSD, which is desirable for oral administration of drugs with low toxicity and is effective in treating and recovering CPVSD by causing regression and retrograde of prescribed lesions.

Pentosan polysulfate (PPS) is a highly sulfated semisynthetic polysaccharide having a molecular weight ranging from about 1,500 Daltons to 6,000 Daltons, depending on the form of separation. PPS belongs to the same species as heparin and heparinoids, but there are many differences in chemical structure, induction method and physicochemical properties between PPS and heparin. Heparin is usually isolated from mammalian tissues such as bovine muscle, pig muscle, liver, and intestine, but PPS is a semisynthetic compound, and its polysaccharide backbone, xylan, is extracted from the bark of beech or other plant material. Sulfating agents such as sulfyl trichloride and acid. After sulfation, PPS is usually treated with sodium hydroxide to give sodium salts.

As can be seen from the formula

Heparin is a sulfated polymer in which two sugar monomers, (D) -glucosamine and (D) -glucuronic acid (six tan sugars, both of which are six carbons) are repeated and have an amine functionality on the glucosamine; PPS is a sulfated linear polymer in which the monomer (D) -xylose, which is a pentose consisting of five carbons in its pyranose ring form, is repeated. Heparin's flat polarization rotates to the right and PPS rotates to the left.

Due to the biological nature of PPS, it is used to prolong partial thromboplastin time and prevent cardiac vein thrombosis, but only about 1/15 of the anticoagulant ability of heparin (Wardle, J. Int. Med. Res., 20: 361-370, 1992 overview). PPS has also been described as useful in the treatment of urinary tract infections and cystitis between cell tissues (see US Pat. No. 5,180,715) and angiosteroidal steroid combinations, capillaries, cells or membrane effusions (see US Pat. No. 4,820,693). have.

Some researchers have described that PPS inhibits smooth muscle cell proliferation and reduces hyperlipidemia, and on such subjects, PPS restricts atherosclerotic plaque formation, inhibits glomerular interstitial cell proliferation, It has been suggested that it may be prophylactically useful in the prevention of glomerulosclerosis (Paul et al., Thromb. Res., 46: 793-801, 1987; Wardle, supra). However, no one previously focused on the site of injury of CPVSD (as a response to inhibition of cell proliferation), such as atherosclerosis, and although PPS was not considered in this context, it is possible to stop or alleviate vascular damage. It is described. Moreover, none of the proposals in the previous field of the viable utility of PPS in impaired disease was supported by effective and scientific efficacy data generated in normal animals, but instead was based on in vitro studies of animal tissues. The study does not show efficacy in vivo.

Recently, there has been literature describing the use of PPS to prevent the development of fibrosis and scarring (see US Pat. No. 5,605,938 to Roufa et al.), But such literature has been shown to prevent fibroblast development in the skin and associated tissues. It only describes suppression, but does not describe pathologically or pathologically different disorders of smooth muscle cells.

Summary of the Invention

It is an object of the present invention to provide a method of treating CPVSD, which not only stops the progression of the disease but also substantially treats the process and causes of existing damage or lesions. It is a further object of the present invention to provide a method of treatment using commercially available pharmaceutical agents which can be administered by routine methods. It is non-toxic and does not cause serious side effects and is very effective in treating CPVSD.

Briefly described, the present invention will be clarified below as to a method of treatment for stopping a patient's progression in a mammalian patient suffering from CPVSD and alleviating or alleviating damage or fibrosis lesions already formed in a damaged organ or pulmonary system. The method comprises administering to the patient a pharmaceutical composition containing a therapeutically effective amount of pentosan polysulfate for its pharmaceutically acceptable salt or vascular injury disease. Oral administration of PPS is preferably administered, for example, in tablet, capsule or liquid form.

Figure 1 shows the quantification of α ₁ IV collagen mRNA of glomeruli 1/10 obtained from normal mice at 5 weeks of age (described in Example 1 below) by competitive PCR:

a) gel stained with ethidium bromide after reaction diagram and PCR amplification (top panel); And

b) Graph depicting the ratio of variant collagen cDNA / glomeruli to the amount of variant cDNA added to each of nine test tubes containing PCR reagents (bottom panel).

2 shows the following:

a) PAS-stained kidney cross section through two nephrectomy specimens with kidney cancer (A-normal glomerular histology; B-marked sclerosis) in the top panel.

b) antibodies to type IV collagen, immunofluorescence microscopy in the same kidney in the middle panel (C-D); And

c) In the bottom panel (E), the bar graph represents the sclerosis index at the same kidney; α ₁ Ⅳ collagen cDNA was 50 microscopically dissected glomeruli (arc value: 145 ± 22 vs 1046 ± 74 × 10 ^-4 atomol / glomerule).

FIG. 3 is a bar graph showing the sclerosis index in the kidney, the relative cell number of glomeruli and α ₁ IV type collagen cDNA level in comparison with 5 patients without glomerulosclerosis and 5 patients with sclerosis.

FIG. 4 shows α ₂ / α ₃ IV in patients with membranous glomerulonephritis (MN) and diabetic nephropathy (DM), nephrectomy (NX GS), and without glomerulosclerosis (NX NI). Type collagen mRNA ratio is shown as a bar graph.

FIG. 5 shows PPS for DNA synthesis in normal glomerular stromal cells by plotting the concentration of tritiated cpm / 10 ³ cells vs. PPS sodium at μg / ml as determined by tritiated thymidine insertion (24 hour incubation) The effect of sodium is shown in a bar graph.

FIG. 6 shows in bar graph the effect of PPS sodium on cell growth by plotting the concentration of added PPS sodium at 3 μg / ml versus the number of cells after 3 days of culture in normal glomerular stromal cells.

7 is a bar graph comparing the effect of PPS sodium and heparin (untreated control) on cell growth in normal glomerular stromal cells after 3 and 5 days of culture.

8 is a graph showing the normal glomerular interstitial cell proliferation several times in comparison with cells cultured with serum and PPS sodium and control cells cultured only with serum.

FIG. 9 shows normal glomerular interstitial cell layers exposed to PPS sodium (100 μg / ml) and reverse-transcribed for several time periods to form α ₁ IV and α ₁ I collagen mRNA, collagenase (metalloproteinase) 72 kDa. And mRNA levels showing increases and decreases in 92 kDa mRNA, growth factor TGF-β mRNA and cellular protein β-actin mRNA levels.

FIG. 10 shows relatively close examination of glomeruli in GH-transformed mice treated with PPS sodium treated beverages for 10-12 weeks and glomeruli in control GH mice treated with untreated beverages, as determined by competitive PCR. In comparison, the ratio of α ₁ IV type collagen / GAPDH is shown in a bar graph.

11 shows the abdominal aorta and PPS sodium (Elmiron) of Watanabe rabbits euthanized in the untreated control group. A cross-sectional view of the abdominal aorta of Watanabe rabbits in the group treated subcutaneously).

FIG. 12 is a bar graph comparing the endothelial cross-sectional view of the various branches of the aorta of the Watanabe rabbit with only a high cholesterol diet and the endothelial cross-sectional view of the different branches of the aorta of the Watanabe rabbit with a PPS sodium treatment and high cholesterol diet in their drinking water. It was.

FIG. 13 shows the intima ratio of the medial cross-sectional area of several branches of the Watanabe rabbits with only a high cholesterol diet and the medial cross-sectional area of several branches of the aorta of the Watanabe rabbits treated with PPS sodium in the high cholesterol diet and their drinking water. Are compared and shown in a bar graph.

The present invention relates to a therapeutic method for slowly slowing the progression of a disease and alleviating and / or alleviating an already injured lesion in a mammalian patient suffering from chronic progressive vascular injury disease (CPVSD) in an artery such as the aorta and an injured pulse system. will be. The method of treatment for a subject comprises administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt thereof or a vascular injury disease of pentosan polysulfate (PPS).

Diseases that can be treated according to the novel method include, but are not limited to, the following: chronic progressive glomerular disease, including impaired diabetes-induced glomerulosclerosis; Arterial injury due to atherosclerosis, including atherosclerosis; Progressive kidney transplant failure due to damage between cellular tissues following kidney transplantation; Occlusion due to injury anastomosis used to enter the vessels in patients with end-stage renal disease treated with hemodialysis; Other chronic impaired small vessel disease (eg, patients with high blood pressure); Relapse of stenosis due to injury in patients with coronary bypass surgery; And diabetic retinopathy.

Because of the prevalence and malignant nature of the disease, its particular importance is in the treatment of alleviating existing vascular injuries and lesions by treating or preventing the progression of the disease for chronic atherosclerotic injury pathology. For example, in accordance with the present invention, PPS administration can stop or treat the progression of atherosclerosis in a number of blood vessels, which alleviate and / or alleviate diseased arteriovascular damage by plaques of atherosclerosis. In general, an increase in endothelial cross-sectional area may cause large amounts of blood to flow through the vascular lumen.

As used herein, the term “therapeutic effective amount for vascular injury disease” refers to the amount of PPS or salt thereof added to a pharmaceutical composition that is effective when administered once or more per day for the time specified in stopping or treating progressive symptoms of CPVSD. It was. In human patients, the total daily dose is about 5 to about 30 mg / kg of the patient's body weight or about 350 to about 2000 mg per day in an adult patient and about 500 to about 1500 mg of PPS or PPS salt. It is preferred that the daily dose is equally divided into 1 to 4 times and is effective in achieving the therapeutic purpose of treating and recovering CPVSD. For smaller mammals, the range of dosage can be adjusted downward depending on body weight, species and type of symptoms.

A preferred embodiment of the novel method of treatment is to administer to the patient a pharmaceutical composition comprising an effective amount of PPS and at least a pharmaceutically acceptable inactive ingredient. The composition may be any standard pharmaceutical dosage form, but oral dosage forms are preferred.

Dosage forms for oral transport include tablets, coated tablets, coated capsules or coated caplets, sustained-release tablets, sustained-release capsules or sustained-release caplets, glycosylated tablets, liquids, elixirs or any other known in the pharmaceutical art. Oral dosage forms.

Pharmaceutically available inactive ingredients are believed to include excipients, binders, solvents and the like that do not interfere with the CPVSD therapeutic activity of PPS. In addition, excipients such as clay or silica earth can be used by adjusting the dosage form as desired.

Ingredients such as excipients and carriers may be necessary to share the desired physical properties of the dosage form. Such physical properties include, for example, release rate, tissue, and size. Examples of excipients and carriers useful in oral dosage forms are waxes such as beeswax, castor wax, glycowax and carnauba wax, methylcellulose, ethylcellulose, carboxymethylcellulose, cellulose-acetate phthalate, hydroxypropylcellulose and hydroxypropylmethyl Cellulose compounds such as cellulose, polyvinyl chloride, polyvinyl pyrrolidone, stearyl alcohol, glycerin monostearate, polymethacrylates, methyl methacrylate and meta methacrylates such as ethylene glycol dimethacrylate, polyethylene glycol and hydrophilic rubber Acrylate compounds.

An amount between about 50 mg and about 300 mg per dosage unit in the composition of the PPS active ingredient of the invention is preferred. The exact dosage administered to each patient will depend on the patient's physical characteristics such as age and weight and the condition treated.

The active pharmaceutical ingredient may be PPS or a pharmaceutically acceptable salt thereof, such as sodium salt. One preferred oral dosage form that can be used in the methods of the invention is Elmiron It is a zilatin capsule (purchased from Baker Norton Pharmaceuticals Inc., Miami, FL), which contains 100 mg of PPS sodium, such as excipients, microcrystalline cellulose and magnesium stearate.

While oral dosage forms are preferred, the methods of treatment of the present invention also include the administration of PPS or salts thereof via parenteral, transdermal, transmucosal pathways or other routes of administration commonly known in the medical and pharmaceutical arts. . Likewise, the compositions of the present invention may comprise PPS in dosage forms to which pharmaceutically acceptable parenteral, transdermal, transmucosal or other conventional excipients and appropriate inert solvents, excipients and additives are added. Many examples of such pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Science (17 th edition (1985)) and other standard texts. Even with the method of administration or the type of pharmaceutical dosage form, the dosage range for the PPS active ingredient is about 5 to about 30 mg / kg or about 350 to 2000 mg of the patient's body weight, although about 500 to 1500 mg is suitable, Doses corresponding to the lower values in the above range will probably be used for parenteral administration.

The pharmaceutical compositions used in the methods of the present invention comprise other active ingredients which are useful in the treatment of CPVSD, such as PPS or PPS salts.

The novel methods allow for the comfort, safe and effective treatment of patients suffering from various forms of CPVSD, which in some embodiments may threaten life or body organs, and in the present methods, low toxicity and low range of side effects Demonstrating pharmaceutical agents can be used to inhibit and / or treat damage lesions that have already been formed to restore the normal vascular openness and function, as well as to stop the progression of chronic vascular injury diseases that have been suffered for a long time.

The following examples include the following:

(a) Descriptions of experiments already published in the medical literature confirm specific competitive PCR (polymerase chain reaction) techniques to quantify damaged collagen mRNA and glomerular related factors, or the production of damaged collagen does not match the relative glomerular cell count. To describe;

(b) The experiment supervised the inventors describing the in vivo and in vitro efficacy of PPS in down-regulating the production of damaged collagen and cell growth elements and up-regulating collagenase activity to degrade the precipitate of damaged collagen. Under the following conditions; And

(c) The experiment was conducted under the supervision of the inventors describing the in vivo efficacy of PPS in recovering atherosclerosis, including substantially reducing the amount and distribution of atherosclerotic plaques in the injured vessel. While these embodiments are not intended, they should be used to practice the invention or to limit the invention in any way in describing the substance, technique or range of administration.

Example 1

Quantification of collagen

Peten et al. Am. As described in J. Physiol., 32: F951-957 (1992), collagen α ₁ IV and α ₂ IV of mouse glomeruli can be quantitatively determined by the following method: from 5 week old normal mice. A standard amount of cDNA and α ₁ IV or α ₂ IV collagen primers corresponding to the mRNA of 1/10 of the glomeruli obtained was obtained from a GeneAmp DNA Amplification Kit (PerkinElmer Citters, Norwalk, CT). Add each of several tubes containing the PCR reagents. Successive dilutions of mutant cDNAs with new restriction sites or deletion sites are added to this mixture before amplification (figure shown in the top panel of FIG. 1). Concentrations of the variants were determined in previous experiments planned for equivalence point brackets (y = 1).

After PCR amplification, the entire reaction mixture was washed with 4% Nusieve: Seakern (3: 1) (FMC Bioproducts, Rockland, Maine) agarose gel with H5 Horizon gel apparatus (Life Technologies). Load it immediately and electrophoresis. DNA bands are identified by ethidium bromide staining and ultraviolet (UV) projection. Photographs were taken with a positive / negative 55 polaroid film (Polaroid, Cambridge, Mass.) (See center panel of FIG. 1). Negative gels are scanned with a competitive one-dimensional laser density meter (Shimadzu; Scientific Instruments, Columbia, MD).

Density measurements and variant bands of wild type were calculated and the ratios in each reaction tube were plotted as a function of the amount of variant template added. For α ₂ IV type collagen variants, the intensity of the measured density band was adjusted by factor 562/479 before showing the variant / wild type band ratio. For α ₂ IV variant bands, density measurements were added before dividing by the wild type band value. Straight lines were drawn by linear regression analysis. The cDNA amount of the wild type sample was calculated as the amount where the variant / wild type band ratio is equal to one. Competitive PCR assays were performed twice or three times.

Example 2

Change in Hardness in Glomeruli

J. Exp. As described above in Med., 176: 1571-1576 (1992), a unidirectional renal extraction sample with renal cell carcinoma was obtained from the patient. The patient has no history of diabetes, hypertension or other systemic diseases associated with glomerular disease. Samples of cortical tissue distant from certain tumors are placed in Carnoy's fixative and placed in methacrylate or paraffin. The cross section is then stained with periodic acid-schiff (PAS). The presence of glomerulosclerosis, defined as swelling of the glomerular interstitial matrix, is characterized by histological examination of the material stained with PAS (top panel in FIG. 2) and immunofluorescence microscopy of frozen sections performed after exposure to antibodies to type IV collagen. Independently evaluated (PHM-12, Silenos, Westbury, NY, USA) (middle panel in FIG. 2).

Competitive PCR analysis was performed as described above in Example 1 where the amount of α ₂ IV (damaged) extracellular matrix collagen was measured. The relative concentrations of glomerular collagen found beforehand in normal persons or sclerosis were measured as shown in the lower panel of FIG. 2.

The relative number of glomerular cells from 5 patients (normal) and 5 patients with sclerosis was compared. As shown in FIG. 3, the difference between groups in the relative number of glomerular cells is not very important (p> 0.8), while the difference in cDNA level of α ₂ Ⅳ collagen is statistically significant (0.01 <p <). 0.025).

Example 3

Of glomeruli and non-normal kidneys of normal kidneys

Relative ratio of collagen mRNA in glomeruli.

Using the methodology described above in Examples 1 and 2, patients with membranous glomerulonephritis (MN) and diabetic nephropathy (DM) and nephrectomy with glomerulosclerosis (NX GS) and patients without glomerulosclerosis (NX NI) Relative proportions of α ₂ / α ₃ Ⅳ collagen mRNA were quantified in the tissue biopsies from patients. As shown in FIG. 4, the ratio of α ₂ / α ₃ Ⅳ collagen mRNA was significantly higher in DM and NS GS than in NX NI. ( ^** P = 0.0002, ^* P = 0.02)

Example 4

In vitro studies on PPS

Study A

Plan of the experiment:

Normal glomerular stromal cells (8) were treated with 20% fetal bovine serum (24 New York, USA) with a 24-well plate at 2-2.5 × 10 ⁴ cells / well concentration (Genk, PGC Scientific Corporation, Guidersburg, Maryland, USA). Basal medium supplemented with Gibco, located in Grand Island. Discard the medium at 24 hours, wash the cells twice with PBS and incubate for 24-72 hours in serum free medium containing 0.1% bovine serum albumin (RIA grade, Sicjma). The medium is replaced with fresh basal medium with 20% fetal bovine serum with and without 5-100 μg / ml PPS and compared with standard heparin (100 μg / ml). Trypsinized cells in paired wells and elzone at +3 and +5 days Counting is performed with a cell counter (Particle Data Inc., Elmhurst, Ill.). In parallel wells, 1 μCi / well of [ ³ H] thymidine ([methyl- ³ H] thymidine); (2.0 Ci / mM; DuPont NEN, Boston, Mass.) Is added to allow thymidine binding. Count on days 1 and 3

result :

The maximum dose-response at 1 day reached a stable level at 50 μg / ml (FIG. 5), but the maximum inhibitory response at 3 days appeared at 25 μg / ml (FIG. 6).

When PPS was not added (control), heparin (100 μg / ml), and PPS (100 μg / ml), PPS treatment showed approximately twice as much efficacy as natural heparin (FIG. 7). Reproduction of the reaction is possible (the error bars are very short).

The summary graph (FIG. 8) compares the effect of adding PPS to serum and control cells exposed to serum only.

Study b

Normal glomerular stromal cell layers are exposed to PPS (100 μg / ml) for several periods of time and reverse-transcribed to measure mRNA levels with selected molecules on day 1 and compared with those on days 3 and 5 (see FIG. 9). There was no change in type IV collagen mRNA, type I collagen mRNA was significantly decreased, TGF-β mRNA was dropped to 50% and 92 kDa enzyme activity was increased by more than 50%. The control is β-actin and is consistent with no change and no proliferation in treated cells.

Example 5

Studies on GH Transgenic Mice

Plan of the experiment:

Twelve 6-week-old GH transgenic mice were identified by PCR analysis of material washed-extracted from tail tissue biopsies using primers specific for bovine growth hormone cDNA that did not cross-react with the mouse GH sequence.

Six GH mice were treated with oral PPS sodium (Elmiron). Baker Norton Pharmacuticals, Inc.) was added to drinking water for 10-12 weeks, and six older GH mice consumed untreated water for the same period. The amount of PPS sodium in the beverage is about 100 mg / kg body weight of the experimental animal.

Glomerular Isolation and Normal Site Reverse Transcription:

The glomeruli are separated by brown rice dissection in the presence of an RNase inhibitor. Saline is perfused to the left kidney and the collagenase solution containing the water-soluble RNase inhibitor is perfused to the saline of the left kidney. The lower pole is removed prior to perfusion with collagenase and flash frozen in dry ice for zymography. After cleavage with collagenase, 40-60 glomeruli are isolated by reverse transcription (RT) at 4 ° C. in the presence of a vanadyl ribonucleoside complex. Normal site RT is performed as above, but glomeruli are freeze-thawed once in acetone dry ice and 2% tryptone and 4 units / μL human placenta RNase inhibitor (Indianapolis, Indiana, USA) prior to addition of RT composition. Sonicated for 5 minutes in the presence of Zöllinger Mannheim. Samples are refrigerated with a Micro Ultrasonic Cell Disrupter (available from Contes, Vineland, NJ) during sonication.

Standard and Competitive PCR Assays:

Primers for mRNA of mouse α ₁ IV and type I collagen, α smooth muscle cell actin, β-actin, lamidine B1, tenascin, 95 kDa metalloproteinase and 72 kDa metalloproteinase Primers for bovine growth hormone genomic DNA were synthesized on PCR-mate (Applied Biosystems, Foster City, Calif.). Size and restriction enzyme analysis distinguish each amplified product. Specificity of primers for mRNA can be determined by excluding reverse transcription enzymes. PCR uses a Gene Amp DNA Amplification Kit (purchased from PerkinElmer Citters, Norwalk, Connecticut). CDNA derived from a pool of 40-60 mouse glomeruli is first analyzed by standard PCR using the log-linear portion of PCR amplification. This allows non-quantitative determination of mRNA levels. Thereafter, α ₁ IV collagen (and the ratio of α ₁ IV collagen to GAPDH enzyme, calculated by standardizing data between experimental animals), PDGF-B, α smooth muscle cell actin, β-actin and laminin B1 cDNAs are subjected to competitive PCR analysis using measurements by the construction of small deletions or cDNA variants of each molecule with new restriction enzyme sites. PCR product analysis is Quantity One This is done using a PDI densitometer loaded with image analysis software. Competitive PCR analysis is performed twice and three times.

result :

As shown in FIG. 10, the type IV collagen / GAPDH ratio was less than half in the group treated with oral PPS sodium than in the non-treated (control) mouse. This difference suggests that there is a very small amount of damaged-type collagen in the glomeruli of the treated animals compared to the untreated animals, which is evidenced by histological examination and immunofluorescence microscopy.

Example 6

Study on Watanabe Rabbit

Watanabe rabbits (these varieties of rabbits are professionally known as Watanabe hereditary fat and hypertension (WHHL)) serve as animal models of innate endogenous cholesterol hyperemia. It is fully expressed in the homozygous state, partially expressed in the heterozygous state, and has characteristics attributed to single-gene deletions. Homozygous Watanabe rabbits have a concentration of 8-14 times more serum cholesterol than normal Japanese white rabbits.

Watanabe rabbits have a very high incidence of atherosclerotic plaques, especially in the aorta. The rate of progression and exacerbation of atherosclerosis can be increased by feeding a high cholesterol diet.

The following two studies deal with anti-atherosclerosis activity of PPS in Watanabe rabbits.

Study A: Subcutaneous Administration Effect of PPS

Twelve Watanabe rabbits are divided into two groups of six each (group A and group B) and fed a high cholesterol diet (0.5% cholesterol). Animals in group A are subcutaneously administered normal saline daily, whereas animals in group B are treated with PPS sodium (Elmiron 10 mg / kg is administered subcutaneously daily.

Four of the PPS-treated animals (group B) died before the study was completed. One died at 22 and three died between 80 and 86 days.

On day 89, animals of group A and the remaining two of group B are euthanized and necropsied to examine fragments from multiple aortic branches.

result :

As shown in Table I below, much less plaque deposits were found in animals in group B, and the ratio of smooth muscle layer to plaque was much higher when all aortic cross section tests were compared to control rabbits in group A (6.8). About twice as high). It can be seen visually in the picture shown in FIG. Abdominal aortic sections in control animals show highly developed forms of atherosclerotic plaques in subcutaneous sections. The abdominal aortic cross section of the PPS sodium treated animals showed almost no signs of plaque, despite being a treatment group fed a high cholesterol diet as in the control group.

Study B: Oral Administration of PPS

Twenty Watanabe rabbits are divided into four groups, five each from Groups A to D. All rabbits are fed the same high cholesterol diet (0.5% cholesterol). Animals in groups A and B are given untreated water, while animals in groups C and D are 0.5 mg / ml of PPS sodium (Elmiron Give water containing). Based on previous studies on water consumed by animals, the total daily dose of PPS sodium consumed by each animal in the treatment group is about 30 mg / kg.

Two of the treated rabbits died of apparent tumors on day 4 and 11 of the study, independent of PPS, respectively.

Animals in groups A and C were euthanized on day 50 of the study and necropsied and their aorta examined. Visually noticeable differences were observed between the group A animals (control) and the group C animals (treatment), with less developed atherosclerosis.

Rabbits in groups B and D were euthanized on day 64 of the study. The aorta of these groups was examined histologically and cross-sectional views of the intima and middle layer sites in various aortic branches were measured respectively.

FIG. 12 is a bar graph reflecting the mean value of the intima site measured in cross-sections obtained from each of the various aortic branches of the control group (group B) rabbits and treatment group (group D). FIG. 12 shows that aortic branching of each of the intima sites shows improvement in disease in treated animals rather than untreated animals, indicating that there are virtually fewer atherosclerotic lesions and plaque deposits in the vasculature of the treated group.

FIG. 13 shows the mean value of the ratio of the intima to the central region in the cross-sectional view of the aorta obtained from rabbits of groups B and D as described in FIG. Reflecting the relationship between the amount of plaque deposited on the vessel wall (increased deposits in the section of the intima), this ratio is higher in all aortic branches of treated rabbits (group D) than in untreated animals (group B). Appears low.

The aforementioned data, carried out by scientifically confirmed experiments, demonstrate the PPS effect of increasing the activity of collagenase, while reducing the production of excess extracellular matrix collagen and the production of specific cell growth factors. These effects indicate that PPS is very effective in the recovery of clinical treatment techniques and CPVSD, especially atherosclerosis and atherosclerosis.

Thus, it can be seen that the present invention provides methods and compositions that achieve the various objects of the present invention, and provides methods and compositions that are well adapted to the conditions of actual use.

It is to be understood that the invention may make various feasible embodiments, and that the embodiments described above may be modified and that all of the materials described herein have been described by way of example and not of limitation.

Novel inventions intended to be protected by patent rights are set forth in the following claims.

Claims (16)

A pharmaceutical composition containing a therapeutically effective amount of pentosan polysulfate (PPS) or a pharmaceutically acceptable salt thereof, to prevent the progression of chronic progressive vascular injury disease (CPVSD) and to lessen or alleviate the damage lesions already formed. A method of treating a mammalian patient suffering from CPVSD, which narrows the lumen of the damaged vasculature and slows dilation, comprising administering to the patient. The method of claim 1, wherein the damaged vasculature is an artery. The method of claim 2, wherein the artery is an aorta or a plurality of branches thereof. 3. The method of claim 2, wherein the disease is in the form of atherosclerosis by injury and the injury process of atherosclerosis is recovered by the treatment method. The method of claim 4, wherein the atherosclerosis form is atherosclerosis and the injury comprises an arterial wall damaged by atherosclerotic plaque. The pharmaceutical composition of claim 1, wherein said pharmaceutical composition is provided in an amount sufficient to provide about 5 to about 30 mg / kg patient weight or about 350 to about 2,000 mg of PPS or a pharmaceutically acceptable salt thereof in a total daily dosage. A method of treatment comprising administering to a patient. 7. The method of claim 6, wherein the daily dose is about 500 to about 1,500 mg. 7. The method of claim 6, wherein the daily dose is divided into one to four doses to administer the same amount. The method of claim 1, wherein the pharmaceutical composition is in oral dosage form. The method of claim 9, wherein the dosage form is selected from conventional tablets, sustained release tablets, coated tablets, caplets, sugar-containing tablets, liquids and elixirs. The method of claim 9, wherein the dosage form comprises at least one of pharmaceutically acceptable inactive ingredients. The method of claim 11, wherein the inactive ingredient is a filler, binder, solvent, excipient or carrier. 10. The method of claim 9, wherein said dosage form contains about 50 to about 300 mg per PPS or pharmaceutically acceptable salt unit thereof. The method of claim 1, wherein the pharmaceutically acceptable salt is a sodium salt. 15. The method of claim 14, wherein the composition is in the form of a gelatin capsule containing PPS sodium, microcrystalline cellulose and magnesium stearate. The method of claim 1, wherein the patient is a human.